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Characterization of pore opening relevant residues in TM3 and TM4 domains of Orai1
ANDOVA, Ana-Marija
Calcium (Ca2+) ions play a crucial role in almost every aspect of cellular life. The most prominent calcium entry pathway into the cell is the calcium release-activated calcium (CRAC) channel, composed of the Orai1 protein, and the stromal interaction molecule STIM1. The channel is activated through conformational changes upon STIM1 coupling to the C-terminus of Orai1 protein following store depletion, which in turn allows Ca2+ influx into the cell. The abnormal function of the CRAC channel caused by mutations gives rise to distinct pathologies. Since it has not yet been elucidated how the signal propagation moves to the pore upon coupling, this thesis dives into its investigation by focusing on characterizing the TM3 and TM4 domains and their importance in leading to an open permissive conformation of the channel. The pivotal foundation for the creation of novel strategies in the modulation of the Orai1 function lies with the understanding of the dynamics of the Orai1 pore opening.
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Cellular and molecular mechanisms of TRPC5 regulation
Ptáková, Alexandra ; Vlachová, Viktorie (advisor) ; Tureček, Rostislav (referee)
The TRPC5 ion channel is a molecular cold detector involved in the development of neuro- pathic and inflammatory pain in the peripheral nervous system. Its possible involvement in the mechanisms of the development of cold allodynia emerging as a side effect of chemotherapeutic treatment has not yet been investigated. The activation properties of the human TRPC5 receptor expressed in HEK293T cells in the context of exposure to low temperatures and oxaliplatin - widely used cytostatic drug, which side effects often manifest as a peripheral neuropathy trig- gered or enhanced by cold temperatures, were investigated using patch clamp electrophysiology and calcium imaging. Molecular modelling was used to explore possible mechanisms of action of oxaliplatin and lysophosphatidylcholine 18:1 whose levels are increased after oxaliplatin treatment. Low temperature (5 řC) decreased the amplitude of agonist-evoked membrane cur- rent responses and slowed down their deactivation. Analysis of single-channel recordings re- vealed increased open probability of the channel as well as prolonged mean open dwell time and reduced mean closed dwell time upon cooling. The highest temperature sensitivity of the gating of the channel was approximately between 16-11 řC with the corresponding value of the temperature...
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The effect of the mast cell activation on the microtubule organisation
Hájková, Zuzana ; Dráber, Pavel (advisor) ; Binarová, Pavla (referee)
The activation of bone marrow-derived mast cells (BMMCs) induces a number of cell processes such as degranulation, proliferation and cytoskeleton rearrangements. Although microtubules are important in these processes, molecular mechanisms that control changes in microtubule organisation during cell activation are unknown. Activation of BMMCs can be achieved in several ways. Under physiological conditions, the aggregation of IgE receptors (FcRI) on the surface of BMMCs leads to the initiation of specific signaling pathways. Cells can be also activated nonspecifically by a tyrosine phosphatase inhibitor pervanadate, or by thapsigargin that inhibits Ca2+ ATPase pumps located on the endoplasmic reticulum. In this diploma thesis it was found out that rapid morphological changes can be monitored when BMMC are immobilised on the fibronectin before their activation. It was proved that specific and nonspecific activation events lead to microtubule reorganization, as well as to generation of a large number of microtubule-dependent protrusions. In the course of FcRI aggregation, generation of microtubule protrusions depends on the activity of Src family protein tyrosine kinases and on the intracellular Ca2+ concentration. STIM1, an endoplasmic reticulum Ca2+ sensor, which participates in the activation of...
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